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MICROBIOLOGY STANDARD OPERATING PROCEDURE
MICROSCOPY OF SPUTUM OR GASTRIC ASPIRATES TO IDENTIFY ACID FAST BACILLI
Document number / version:
Reviewed and approved by:
Replaces document:
Date of original:
Applies to:
Microbiology laboratory
Modified by:
1
Sep-2005
Date of revision:
Date for review:
Aim
To describe the procedure to presumptively identify Mycobacterium tuberculosis (TB) infection by
detection of acid fast bacilli (AFB) in gastric aspirate or sputum specimens.
2
Principle
Tuberculosis is caused by the organism Mycobacterium tuberculosis. The lung is the most frequent
site of infection. Infection can be presumptively identified by detection of acid fast bacilli in sputum
or gastric aspirate specimens. Early morning gastric aspirates consist of sputum swallowed
overnight and are useful in young children who cannot easily cough up sputum.
Ziehl-Neelsen (ZN) stain renders mycobacteria pink as a result of the mycolic acid in their cell wall.
Most other bacteria have a lower fat content and hence the carbol fuchsin is washed away by the
acid-alcohol, resulting in their being stained blue by the counterstain (methylene blue). Hence,
mycobacteria are referred to as being “acid fast”.
Page 1 of 5
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MICROBIOLOGY STANDARD OPERATING PROCEDURE
MICROSCOPY OF SPUTUM OR GASTRIC ASPIRATES TO IDENTIFY ACID FAST BACILLI
Document number / version:
3
Method
3.1 Specimen collection
Specimens should be collected into wide-mouthed leak-proof sterile plastic pots.
Three sequential day gastric aspirates (young children) or sputum specimens (older children)
should be obtained for investigation of suspected TB infection.
3.2 Specimen transport and storage
Specimens should ideally be stored and transported in sealed plastic bags. Laboratory processing
should occur as soon as possible after specimen collection. Specimens should be refrigerated if
delays in processing over two hours are unavoidable.
3.3 Specimen processing
3.3.1 Reception
Log the specimen in the appropriate specimen book and assign a specimen number.
The specimen should also be logged in for molecular detection of Mycobacterium tuberculosis
(GeneXpert, SOP MOL-002).
3.3.2 Microscopic examination
Preparation of the smear must be done in the Class II biosafety cabinet.
Label a clean glass slide with the patient code specimen number.
Using a bamboo/wooden stick, select a blood-stained or purulent portion of the specimen.
Smear the specimen in the centre of the slide (2cm x 1cm).
Discard the wooden stick into the 1% Virkon discard container.
Leave the smears in the cabinet to dry for 15-30 minutes.
Follow the ZN stain procedure (SOP MID-001)
Examine systematically using the 100x oil objective lens.
4
Interpretation
Acid fast bacilli (AFB) are slender rods which vary from 0.5-10 µm in length and stain red. Some
may appear beaded. TB bacilli may occur singly, as v-shaped forms, or as clumps of bacilli.
Page 2 of 5
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MICROBIOLOGY STANDARD OPERATING PROCEDURE
MICROSCOPY OF SPUTUM OR GASTRIC ASPIRATES TO IDENTIFY ACID FAST BACILLI
Document number / version:
All other organisms and background material stain blue (if methylene blue counterstain is used).
4.1 Reporting
Report the absence or quantity of AFB seen as outlined in the table.
Number of AFB
(x100)
Report
No AFB in 100 fields
AFB negative
1 - 9 AFB in 100 fields
+/- AFB
(report number seen)
10 - 99 AFB in 100 field
1+ AFB
1 - 10 AFB per field
(check 50 fields)
2+ AFB
>10 AFB per field
(check 20 fields)
3+ AFB
5
Quality assurance
ZN stain reagents should be quality controlled according to SOP MID-001.
6
Limitations
Spores and artefacts may stain with Ziehl-Neelsen’s stain and appear as positive to untrained
eyes.
Poor quality specimens may yield false negative results.
7
References
1. Health Protection Agency, UK SOP B40: Investigation of Specimens for Mycobacterium
species (Issue 6.0; August 2012).
2. Cheesbrough, M. District Laboratory Practice in Tropical Countries, Part 2. 2 nd Edition
Update (2006). Cambridge University Press.
3. Standard Operating Procedures from LOMWRU, SMRU and AHC.
Page 3 of 5
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MICROBIOLOGY STANDARD OPERATING PROCEDURE
MICROSCOPY OF SPUTUM OR GASTRIC ASPIRATES TO IDENTIFY ACID FAST BACILLI
Document number / version:
8
Synopsis / Bench aid
Not applicable
Page 4 of 5
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MICROBIOLOGY STANDARD OPERATING PROCEDURE
MICROSCOPY OF SPUTUM OR GASTRIC ASPIRATES TO IDENTIFY ACID FAST BACILLI
Document number / version:
9
Risk assessment
COSHH risk assessment - University of Oxford COSHH Assessment Form
Description of procedure
Microscopy of sputum or gastric aspirates to
identify acid fast bacilli
Substances used
ZN stain reagents (carbol fuchsin, 3% hydrochloric
acid in isopropyl alcohol, methylene blue)
Quantities of chemicals used
Frequency of SOP use
Small
Daily
Hazards identified
Could a less hazardous substance be
1. Potentially infectious material in sample
used instead?
2. Stain reagents can cause burns, are
No
harmful by inhalation, skin contact and
ingestion
What measures have you taken to control risk?
1. Training in good laboratory practices (GLP)
2. Appropriate PPE (lab coat, gloves, eye protection)
3. Use of biosafety cabinet for preparation of slides
3. Small amounts of stain are in use only (stocks kept in a closed chemical cupboard)
4. Laboratory is well ventilated
Checks on control measures
Observation and supervision by senior staff
Is health surveillance required?
Training requirements:
No
GLP
Emergency procedures:
Waste disposal procedures:
1. Report all incidents to Safety Adviser
1. Sharps discarded into appropriate rigid
2. Use eyewash for splashes
containers for incineration
3. Clean up spills using 1% Virkon or
2. Infectious waste discarded into autoclave bags
chemical spill kit
or 1% Virkon solution prior to autoclaving and
subsequent incineration
3. All stains are washed down the sink with
copious amounts of water
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